Among biological materials, collagen, particularly type I collagen, is a major component of various connective tissues including bone. The replacement of human tissues with human- or animal-derived tissues such as skin or bone grafts results in the improvement of the wound healing process because of the presence of collagen. Therefore, the application of collagen-derived products as biomaterials has tremendous impact in biomedicine because of (i) the natural structure of these products as a biological support for cells and scaffold for tissue repair or regeneration, (ii) their biodegradability that obviates removal of implants, and (iii) their biocompatibility. Collagen has been used to design biomaterials such as wound dressings, artificial dermis, bone or tendon substitutes, tissue engineered devices, and injectable materials in plastic surgery .sup.1-5. One of the advantages of using animal collagen, particularly from bovine species, is the facility with which large quantities of pure type I or type I/III collagen can be produced.
The emergence of new viruses and the appearance of new infective diseases require increased vigilance concerning the safety of biologicals, especially since the discovery of transmission of protein such as the prion correlates with infectivity (i.e., bovine spongiform encephalopathy).sup.6. The assessment of the safety of a biological product towards prion is complicated by the lack of a test capable of detecting scrapie-like agents in the starting material. Collagen purified from human sources could also be a vector for the human spongiform encephalopathies.sup.7 and, perhaps, viral diseases (known and unknown).
The scrapie agent is extremely resistant to heat and physical inactivation.sup.8. Prolonged exposure to concentrated NaOH solutions and autoclaving at temperatures above 130.degree. C. can be recommended for routine inactivation and disinfection of scrapie-like agents.sup.9. However, there is still a debate over whether this procedure simply extends the incubation period.sup.10. Furthermore, only NaOH can be applied to collagen because autoclave destroys collagen.
Treatment of collagen by NaOH at similar concentrations and incubation periods (as recommended) have been investigated towards the elimination of other infectious agents such as bacterium or viruses (RNA or DNA viruses). In a basic environment, we have shown indirectly by agarose gel that DNA and RNA are not fragmented enough to implement elimination of infectious disease and transmission.
In another study we have compared the effect of glutaraldehyde treatment as usually used to crosslink collagen products (i.e., Yannas' skin; cardiac biological valves; vessel grafts; and other biological implants). Two methods of treating collagen with glutaraldehyde solution have been tested. One is using water and the other diluted acetic acid as buffer to crosslink collagen. The latter condition is described in Yannas' patent U.S. Pat. No. 4,060,081 on his artificial skin. Many investigators claim that glutaraldehyde treatment of collagen and derived products (i.e., gelatin capsules) can sterilize the final crosslinked products. Using the two methods of glutaraldehyde treatment, our investigation (using agarose gel) shows clearly that DNA and RNA incorporated in our collagen are not completely broken down and subsequently the risk of transmission of viruses and bacteria is most probable.
Other treatments such as 8M urea have been also recommended. Nevertheless, partial breakdown of DNA and RNA was observed. This breakdown is partial enough to offer no warranty of virus-free products.
On the other hand, formic acid (FA), SDS, fluorinated alcohols, and trifluoroacetic acid (TFA) have dramatic effects on the prion (PrP27-30) secondary and tertiary structures which correlates with the inactivation of scrapie infectivity.sup.11-12. There is no suggestion in the art that collagen will resist to those treatments.
As mentioned above, a combination of NaOH and autoclave is currently used to eliminate prion. We have shown that NaOH by itself cannot warrant the breakdown of DNA or RNA. It is further known that autoclaving at about 130.degree. C. destroys collagen. Other procedures making use of severe dehydrothermal treatment are used for crosslinking polymer components. Dehydrothermal treatment also has for effect to sterilize the products as well as crosslinking polymer components. Nobody has investigated as to whether heat sterilization may remove prions while preserving the integrity of collagen-comprising products.
In view of the foregoing, there is a need for a method of making a prion-free collagen-comprising product wherein prion is eliminated while collagen is not substantially denatured beyond a desirable or unavoidable extent.